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Study of the Regulation of NADP-Malic Enzyme (Decarboxylating) and Influence of Stress on this Enzyme
Doubnerová, Veronika ; Ryšlavá, Helena (advisor) ; Wilhelmová, Naďa (referee) ; Kizek, René (referee)
4. Conclusions l. Non-photosynthetic cytosolic isoform of NADP-mďic ilZ-oe (oxaloacetate decarboxylating) (NADp-ME; EC r.l.l.4o) was isolated from tobacco (Nicoriana tabacun L.) leaves. Specific aďiviý of the obtained enz5me preparation was 0.95pmol.miď|.mg l, relďive molecular mass of one subr'nit of tetramer approximatety 67000' pI 5'5 and pH optimum 7.r - 7.4. Kinetic constants of this isoform were determined for L-ma|ate and NADP+ in the presence of various cofactors Mď*, Mo*,co2', Ni2*, similarly the dependencies ofreaction Íates on the ooncentration ofdivalent metal ions were observed- The dependence of reaction rate on Mn2+ concentration wassigmoidal with strong positive cooperativiý and high value of Hill coefficient 7.5. -2r - J. 4. Summarv ofPh.D.Thesis 2. The kinetic meohanism of two-substate reaďion catalyzed by NADP.ME ůomtobacco leaves was determined as ordered sequmtial. The regulation of the NADP-ME purified ůomtobacco leaves occurs predominant|y in the prsence of macmergic compounds (GTP, ATP and ADP), sligbtly by metabolites of glymlysis. Biotic stress caused by potato yirus f G1;Y) (not only strain PWNN but also PWo) increased NADP.ME activiý in N. tabacum L. |eaves, stems and roots during the infeďion. Milder PVYo caused tower increase of NADP-ME aďivity than necrotic isolate of PVYNN....
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Study of selected plant exocyst subunits and its interactors in autophagy pathway.
Rácová, Denisa ; Žárský, Viktor (advisor) ; Wilhelmová, Naďa (referee)
Exocyst is a binding protein complex, which is evolutionary conserved in yeast, animal and in plant cells. It has crucial role in regulation of cell morfogenesis and cell polarity. The function of the exocyst complex is binding of secretoric vesicle to the proper side on plasma membrane in penultimate step of exocytosis. This process is essecial for function and survival of cell. Another process crucial for the cell is autophagy. In plants autophagy plays important role in the responses to nutrient starvation, senescence, abiotic and biotic stress. RabG3b are small GTPases, which have positive role in autophagy. In this work I described the interaction between RabG3b and some of subunits of exocyst complex: Exo70B1, Exo70B2 and Exo84b. I also studied changes in morfogenesis of tonoplast by induction and inhibition of authophagy and induction of anthocyans synthesis in Arabidopsis thaliana.
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Oxidative damage to cellular components after oxidative stress induction by specific herbicides
Kramná, Barbara ; Wilhelmová, Naďa (advisor) ; Ryšlavá, Helena (referee)
Oxidative stress is caused by overproduction and overaccumulation of ROS (reactive oxygen species). This state is responsible for cellular damage during unfavorable environmental conditions such as drought, low temperatures, salinity. In order to directly study oxidative stress at tobacco plants (Nicotiana tabacum cv. Xanthi) I used specific herbicides, MV (methyl viologen) and 3-AT (3- aminotriazole). There were several markers used for monitoring oxidative damage to cellular components: DNA damage detected by a comet assay, lipid peroxidation, carbonylated proteins and modification of activities of antioxidant enzymes CAT (catalase) and APX (ascorbate peroxidase). Fluorescent microscopy documented changes in a redox state of tobacco cells and a specific signal for peroxisomes was observed after treatment with higher concentrations of MV and 3-AT. Application of both herbicides caused significant DNA damage, while they worked in a different concentrations, MV in µM and 3-AT in mM. Another convincing oxidative stress marker for MV was protein carbonylation. The inhibition of antioxidant enzymes CAT and APX was less significant when compared to the effects of 3-AT. Decreasing membrane stability proved to be an universal oxidative stress marker for both herbicides. On the other hand, lipid...
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Antioxidant enzymes response to abiotic stress. Impact of decreased cytokinin level.
Lubovská, Zuzana ; Wilhelmová, Naďa (advisor) ; Prášil, Ilja (referee) ; Kočová, Marie (referee)
Charles University in Prague, Faculty of Science Department of Experimetnal Plant Biology Ph.D. study program: Plant physiology and anatomy Abstract Antioxidant enzymes response to abiotic stress. Impact of decreased cytokinin level. Zuzana Lubovská Supervisor: RNDr. Naďa Wilhelmová, CSc. Supervisor-consultant: RNDr. Helena Štorchová, CSc. Praha, 2015 The response of the antioxidant enzyme system to drought, heat and a combination of these stresses was followed in tobacco plants overexpressing cytokinin oxidase/dehydrogenase1 (CKX1) under the root-specific WRKY6 promoter (W6:CKX1 plants) and under the constitutive 35S promoter (35S:CKX1 plants) and in the corresponding wild type (WT). CKX1 over-producing lines developed a bigger root system, which contributed to their stress tolerance. The high stress tolerance of 35S:CKX1 plants was also associated with a dwarf shoot phenotype and changed leaf morphology. The genes for chloroplastic antioxidant enzymes, stroma ascorbate peroxidase (sAPX), thylakoid ascorbate peroxidase (tAPX) and chloroplastic superoxide dismutase (FeSOD), which are responsible for scavenging of reactive oxygen species produced via electron transfer during photosynthesis, were all strongly transcribed in control conditions. All the tested stresses down-regulated expression of these genes...
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The role of NO in plant senescence
Kramná, Barbara ; Wilhelmová, Naďa (advisor) ; Lhotáková, Zuzana (referee)
1 Abstract Nitric oxide (NO) is a lipophilic free radical gaseous molecule able to readily diffuse through membranes and thus influence the adjacent cells from its source. It belongs among important physiological modulators of a plant life despite of its short life span, which is in most cases, only a few seconds. NO influences plants during their whole life cycle and predominantly acts via a modulation of gene expression or post-translation modifications. An enzymatic production of NO has been documented in apoplast, cytosol and organels such as mitochondria, chloroplasts and peroxisomes, apart from its spontaneous production in reducting conditions. The possible cytoprotective or cytotoxic effects of NO in plant cells depends on its concentration. High concentrations have been proved to be rather cytotoxic. Plant senescence is often accompanied by a vast oxidative damage, which results in high concentrations of ROS, such as O2∙- and H2O2, lipid peroxidation and a decrease of antioxidant enzymes activities. NO has been proved to retard or entirely prevent a senescent phenotype of stress-treated plants. Thus a role as a negative regulator of plant senescence has been proposed for it. The most often method used for NO level modulation in plant tissues is an exogenous aplication of various NO donors. Other...
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Analysis of antioxidant systems, morphological and physiological parameters in two faba bean (Vicia faba L.) cultivars during drought stress
Jelénková, Iva ; Kočová, Marie (advisor) ; Wilhelmová, Naďa (referee)
Knowledge of physiological and morfological mechanisms which are related to drought tolerance is essential in breeding high-yielding and tolerant plants, which would not suffer unfavourable environmental conditions. Drought stress can lead to oxidative damage which causes serious disorders in physiological and biochemical processes in plant cells. Antioxidants can reduce those negative effects. Morphological (dry mass of shoot and roots, height of shoot, number of leaves), physiological (photosynthetic efficiency, pigment content, relative water content-RWC) and antioxidative (catalase-CAT, ascorbate peroxidase-APX, proline) parameters were evaluated in this study using two genotypes of Vicia faba L. differing in drought susceptibility. Drought resistant (Merkur) and sensitive (Piešťanský) genotypes were exposed to 10 days of drought. Activity of CAT and APX of stressed plants rather decreased. Proline content oscillated and no evident trend or significant differences were observed in relation to drought stress. Decrease in RWC was expressed more at susceptible plants, electron-transport chain activity and pigment content were not affected much by drought. Drought susceptible genotype expressed more serious negative effects of drought in morphological parameters, however this genotype was a bit bigger than...
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Isolation and characterization of photosynthetic complexes from photoheterotrophic bacteria
Řádek, Martin ; Wilhelmová, Naďa (referee) ; Kučera, Tomáš (advisor)
Aerobic anoxygenic phototrophs (AAPs) represent a relatively recently discovered unique BChl a-containing bacterial community. These organisms own a fully functional bacterial photosynthetic system. Their photosynthesis is operative only in the presence of oxygen. However, they are not able to grow autotrophically, they require a source of organic carbon for growth and metabolism. I have characterized the photosynthetic apparatus of Erythrobacter sp. NAP1, Roseobacter sp. COL2P and the strains Dor-2m, Dor-vul and Zun-kholbo. BChl a performs the function of the main light-harvesting pigment. Spheroidenone is the major carotenoid of the strains COL2P, Dor-2m, Dor-vul and Zun-kholbo. The NAP1 strain contains "non-photosynthetic" erythroxanthin sulfate, which is bound neither to the reaction center nor to the light-harvesting complex. Bacteriorubixanthinal, zeaxanthin and erythroxanthin are the main photosynthetic carotenoids of NAP1, coupled to the light- harvesting system. This system is composed of inner LH1 complex and reaction center, with no peripheral LH2 complexes. The photosynthetic unit size, in comparison with a typical anaerobic phototroph Rhodobacter sphaeroides, is limited due to the absence of LH2 antenna. The purified LH1-RC complex isolated from NAP1 is relatively thermostable,...
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